U.S. patent number 8,394,372 [Application Number 11/992,061] was granted by the patent office on 2013-03-12 for stabilized protease composition.
This patent grant is currently assigned to Trobio AB. The grantee listed for this patent is Hans Ageland, Lars-Olov Andersson. Invention is credited to Hans Ageland, Lars-Olov Andersson.
United States Patent |
8,394,372 |
Andersson , et al. |
March 12, 2013 |
**Please see images for:
( Certificate of Correction ) ** |
Stabilized protease composition
Abstract
A composition is provided, which comprises a serine protease; a
reversible inhibitor of said serine protease; and a stabilizing
agent M having the formula I: ##STR00001## Also provided are uses
of the composition as a medicament, and other uses and methods
employing its various properties.
Inventors: |
Andersson; Lars-Olov (Nacka,
SE), Ageland; Hans (Saltsjo-Boo, SE) |
Applicant: |
Name |
City |
State |
Country |
Type |
Andersson; Lars-Olov
Ageland; Hans |
Nacka
Saltsjo-Boo |
N/A
N/A |
SE
SE |
|
|
Assignee: |
Trobio AB (Nacka,
SE)
|
Family
ID: |
34926632 |
Appl.
No.: |
11/992,061 |
Filed: |
September 21, 2005 |
PCT
Filed: |
September 21, 2005 |
PCT No.: |
PCT/SE2005/001391 |
371(c)(1),(2),(4) Date: |
March 14, 2008 |
PCT
Pub. No.: |
WO2007/035143 |
PCT
Pub. Date: |
March 29, 2007 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20090136474 A1 |
May 28, 2009 |
|
Current U.S.
Class: |
424/94.3;
435/188; 435/214 |
Current CPC
Class: |
A61K
31/13 (20130101); C07D 295/13 (20130101); A61K
38/4833 (20130101); C12N 9/96 (20130101); A61K
31/5377 (20130101); A61P 7/00 (20180101); A61K
38/484 (20130101); A61L 31/047 (20130101); C07D
295/088 (20130101); A61K 45/06 (20130101); A61L
15/32 (20130101); A61K 47/18 (20130101); A61K
47/22 (20130101); A61K 31/13 (20130101); A61K
2300/00 (20130101); A61K 31/5377 (20130101); A61K
2300/00 (20130101); A61K 38/4833 (20130101); A61K
2300/00 (20130101); A61K 38/484 (20130101); A61K
2300/00 (20130101) |
Current International
Class: |
A61K
38/54 (20060101); C12N 9/96 (20060101) |
Field of
Search: |
;435/194,188,212,213,214,217,219 ;424/94.3,94.63 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1504580 |
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Jun 2004 |
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CN |
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221700 |
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EP |
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1136084 |
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Sep 2001 |
|
EP |
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1 418 182 |
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May 2004 |
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EP |
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1 637 141 |
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Mar 2006 |
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EP |
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11-147834 |
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Jun 1999 |
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JP |
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2002-3353 |
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Jan 2002 |
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JP |
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2002-516566 |
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Jun 2002 |
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JP |
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2002-526531 |
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Aug 2002 |
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JP |
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2004-509104 |
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Mar 2004 |
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JP |
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2004-517060 |
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Jun 2004 |
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JP |
|
2004-191367 |
|
Jul 2004 |
|
JP |
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WO 98/22619 |
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May 1998 |
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WO |
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WO-99/11658 |
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Mar 1999 |
|
WO |
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WO-00/20394 |
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Apr 2000 |
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WO |
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WO-02/22575 |
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Mar 2002 |
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WO |
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WO-02/37937 |
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May 2002 |
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WO |
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WO-02/100830 |
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Dec 2002 |
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WO |
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WO 03/016347 |
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Feb 2003 |
|
WO |
|
Other References
Sigma Catalog (1998) p. 1876. cited by examiner .
Alrdich Catalog (1996-1997) p. 88. cited by examiner .
Lehninger "Biochemistry" (1975) 2nd eidtion. (Worth Publishers,
Inc.: New York, NY) p. 51-52, 197. cited by examiner .
von der Saal et al. Bioorganic Med. Chem. Lett. (1997) 7(10):
1283-1288. cited by examiner .
Derwent abstract for SU 833985 B Bondarev et al. May 30, 1981
(downloaded from Derwent Feb. 25, 2011). cited by examiner .
Markwardt et al. European J. Biochem. (1968) 6: 502-506. cited by
examiner .
English machine translation for JP 2004191367 downloaded from the
JPO Mar. 20, 2012. cited by examiner .
Derwent abstract for for JP 2004191367 downloaded from WEST Mar.
20, 2012. cited by examiner .
Chinese Office Action Issued in Application No. 200580051655.0 on
Apr. 22, 2010. cited by applicant .
Kikugawa Norihiro: "Thrombin reagent and test reagent kit" CA,
2004, XP002337251. cited by applicant .
Nakamura et al., "Characterization of p-aminobenzamidine-based
sorbent and its use for high-performance affinity chromatography of
trypsin-like proteases", Journal of Chromatography A, 1009, 2003,
pp. 133-139. cited by applicant .
Turner et al., "p-Amidino Esters as Irreversible Inhibitors of
Factors IXa and Xa and Thrombin", Biochemistry, 1986, 25, pp.
4929-4935, XP000608122. cited by applicant .
Yang et al., "Synthesis of Novel Biodegradable Cationic Polymer:
N,N-Diethylethylenediamine Polyurethane as a Gene Carrier",
Biomacromolecules 2004, vol. 5, pp. 1926-1932. cited by applicant
.
English language Japanese Office Action, dated Aug. 23, 2011, for
Japanese Application No. 2008-532186. cited by applicant.
|
Primary Examiner: Hanley; Susan
Attorney, Agent or Firm: Birch, Stewart, Kolasch &
Birch, LLP
Claims
The invention claimed is:
1. A stabilized serine protease composition comprisingan aqueous
solution of a) thrombin; b) a reversible inhibitor of thrombin
which exhibits a K.sub.i value of between 0.01 and 2 mM; and c) a
stabilizing agent M, wherein M is 3-(N-morpholino)propane sulfonic
acid and M is present in a concentration of 0.20 M.
2. The composition according to claim 1, in which said thrombin is
human thrombin.
3. The composition according to claim 1, which further comprises a
viscous and adhesive polysaccharide.
4. The composition according to claim 3, in which the
polysaccharide is selected from starch, its derivatives, cellulose,
its derivatives, and mixtures thereof.
5. The composition according to claim 4, in which the
polysaccharide is selected from carboxymethyl cellulose, ethyl
hydroxyethyl cellulose and mixtures thereof.
6. The composition according to claim 3, in which the
polysaccharide is carboxymethyl chitosan.
7. The composition according to claim 3, in which said
polysaccharide is present in a concentration of 0.1-5%.
8. A composition according to claim 7, in which said polysaccharide
is present in a concentration of 1.0-2.0%.
9. The composition according to claim 1, in which thrombin is
present in a concentration of 0.001-2 mg/ml.
10. The composition according to claim 1, in which the
concentration of thrombin is 5-3500 activity units/ml.
11. The composition according to claim 10, in which the
concentration of thrombin is 200-1000 activity units/ml.
12. The composition according to claim 10, in which the
concentration of thrombin is 5-20 activity units/ml.
13. The composition according to claim 1, in which said reversible
inhibitor of thrombin is present in a concentration of 0.1-10
mM.
14. The composition according to claim 1, in which the reversible
inhibitor exhibits a K.sub.i value between 0.04 mM and 0.5 mM.
15. The composition according to claim 1, in which the reversible
inhibitor is selected from benzamidine, N,N-diethylethylenediamine,
aminobenzamidine, amidinopyridin, tert-butylamidin, and a compound
having the formula IV: ##STR00009## wherein R.sup.1 is selected
from H, C.sub.1-C.sub.6-alkyl, C.sub.3-C.sub.7-cyclo alkyl, phenyl,
benzyl acetyl and benzoyl; X is selected from oxygen, nitrogen and
sulfur; R.sup.2 and R.sup.3 is each individually selected from H,
halogen, hydroxyl, C.sub.1-C.sub.6-alkyl, C.sub.3-C.sub.7-cyclo
alkyl, C.sub.1-C.sub.6-alkyloxy; and R.sup.4 is selected from H,
C.sub.1-C.sub.6-alkyl, arylalkyl and acyl.
16. The composition according to claim 15, in which the reversible
inhibitor is selected from N,N-diethylethylenediamine,
amidinopyridin, tert-butylamidin, and a compound having the formula
IV: ##STR00010## wherein R.sup.1 is selected from H,
C.sub.1-C.sub.6-alkyl, C.sub.3-C.sub.7-cyclo alkyl, phenyl, benzyl
acetyl and benzoyl; X is selected from oxygen, nitrogen and sulfur;
R.sup.2 and R.sup.3 is each individually selected from H, halogen,
hydroxyl, C.sub.1-C.sub.6-alkyl, C.sub.3-C.sub.7-cyclo alkyl,
C.sub.1-C.sub.6-alkyloxy; and R.sup.4 is selected from H,
C.sub.1-C.sub.6-alkyl, arylalkyl and acyl.
17. The composition according to claim 15, in which the reversible
inhibitor is a compound having the formula IV: ##STR00011## wherein
R.sup.1 is selected from H, C.sub.1-C.sub.6-alkyl,
C.sub.3-C.sub.7-cyclo alkyl, phenyl, benzyl acetyl and benzoyl; X
is selected from oxygen, nitrogen and sulfur; R.sup.2 and R.sup.3
is each individually selected from H, halogen, hydroxyl,
C.sub.1-C.sub.6-alkyl, C.sub.3-C.sub.7-cyclo alkyl,
C.sub.1-C.sub.6-alkyloxy; and R.sup.4 is selected from H,
C.sub.1-C.sub.6-alkyl, arylalkyl and acyl.
18. A stabilized serine protease composition comprising an aqueous
solution of a) thrombin, wherein said thrombin is present in a
concentration of 0.001-2 mg/ml; b) a reversible inhibitor of
thrombin, wherein said reversible inhibitor of thrombin is present
in a concentration of 0.1-10 mM; and c) a stabilizing agent M,
wherein M is 3-(N-morpholino)propanesulfonic acid and M is present
in a concentration of 0.20 M.
Description
FIELD OF THE INVENTION
The present invention relates to an enzyme composition in which the
enzyme is stabilized by certain additives in an inventive
combination. More particularly, the invention concerns a serine
protease composition comprising a reversible inhibitor to the
serine protease and an additional stabilizing agent M as defined
below.
BACKGROUND
Serine proteases are a group of proteolytic enzymes characterized
by having a serine and a histidine residue in their active site.
Many well known enzymes belong to this group, for example trypsin,
kallikrein, thrombin and plasmin. Several of them have found
practical use. Trypsin is used in the leather industry. Thrombin is
used as a haemostatic agent to stop bleeding from wounds. Urokinase
and tissue plasminogen activator, two other serine proteases, are
used clinically as thrombolytic agents in the treatment of acute
myocardial infarction. A number of these enzymes have been used
extensively as research tools, for instance in protein structure
determination. Furthermore, the enzymes are used in various
diagnostic kits.
Common to most of the serine proteases are their limited stability
in solution. This is mainly caused by autodegradation when left in
solution, caused by their property as proteases. This limited
stability is a problem when the material has to be stored in
solution. Commercial serine protease preparations available today
are essentially always in the form of frozen solutions
orlyophilized powders, with obvious drawbacks. The extra time
needed for dissolution of the powder or thawing of the frozen
solution to the correct temperature is the most important issue.
There are, however, other problems with these preparations. For
frozen solutions, there is a need for controlled temperatures
(-20.degree. C.) in all steps from manufacture and transportation
to storage. For lyophilized powders, there is a need for a
reconstitution solution with an acceptable grade of purity and
stability. Also, the material frequently needs to be prepared
aseptically (by mixing of the two parts) in an environment which
may be non-controlled (such as inclement weather or lack of a clean
water supply), and there is a need to verify that the powders have
been properly mixed. These are all major drawbacks of the products
available today, adding to their complexity of use as well as their
cost.
For thrombin, which preferably has to be immediately available for
use in arresting bleeding, the stability problems have forced
manufacturers to use lyophilized thrombin or deep frozen solutions.
These then require a certain amount of time to prepare for use. The
two thrombolytic agents urokinase and tissue plasminogen activator
are sold in the form of lyophilized preparations that have to be
dissolved before use. Since thrombolytic treatment of acute
myocardial infarction has to be started as early as possible after
onset of the infarction, any time delay caused by such preparation
is a problem.
Many efforts have been made to find ways to stabilize the various
serine proteases. For trypsin, which degrades itself fairly
rapidly, a simple and efficient stabilizing agent is the calcium
ion (Sipos T and Merkel J, Biochemistry 9:2766 (1970)). Decreasing
the pH to below 4 is also a method that works with some of the
enzymes, like trypsin and plasmin, but is not feasible with
thrombin, since it is irreversibly inactivated by a pH below 5.
Reversible protease inhibitors can be used, but are less popular,
since they interfere in a detrimental fashion with the action of
the enzyme when they are used by themselves (see below).
For stabilization of tissue plasminogen activator (tPA), addition
of the amino acid arginine is conventionally used. The tPA material
in clinical use today contains arginine as stabilizer.
Also, a lot of effort has been devoted to find ways to stabilize
thrombin solutions. As examples of stabilizing additives, the
following proposals may be mentioned: carboxylic acids in high
concentrations, EDTA, various amino acids, albumin, polymers such
as polyethylene glycol, polyvinyl pyrrolidone and polyvinyl
alcohol, glycerol, various inorganic salts, carbohydrates, gelatin,
collagen.
Japanese patent application JP2004191367 describes a stabilized
thrombin containing test reagent for testing blood coagulation
ability. The test reagent contains thrombin and a thrombin
inhibitor, and may also comprise one or more thrombin stabilizing
compounds selected from calcium ion, an organic acid, a surfactant
and a protein.
WO 02/100830, WO 02/22575, WO 00/20394, WO 99/11658, WO 02/37937
and U.S. Pat. No. 5,409,927 all describe different serine protease
inhibiting compounds and their use in pharmaceutical compositions
for treatment of various disease conditions, such as thrombosis,
wherein inhibition of the corresponding serine proteases is
indicated.
Nakamura et al. (J. Chrom. A, 1009, (2003), 133-139) describe the
use of an immobilized protease inhibitor for affinity
chromatography of trypsin-like proteases.
Turner et al. (Biochemistry, 25, (1986), 4929-4935) describe three
p-amidinophenyl esters that irreversibly inhibit human factor
IXa.
Tsung Fu Yang et al. (Biomacromolecules, 25, (2004), 1926-1932)
describe the synthesis of a cationic polymer,
N,N-diethylethylenediamin polyurethane, for use in gene
delivery.
US patent application 2001/0033837 (corresponding to EP 1 136 084
A1) describes a thrombin preparation containing a non-covalently
bound inhibitor as stabilizer. Furthermore, the inhibitor is
combined with other stabilizing additives, like sugars or
carboxylic acids, which have been previously described in patents
or other publications.
JP 2000300250 describes the stabilization of thrombin solutions by
addition of polyvinyl alcohol, gelatin or polyvinyl pyrrolidone in
different buffer solutions.
In GB 1354761, proteases and amylases are stabilized to various
extents by a number of substances, such as aliphatic alcohols,
carboxylic acids, heterocyclic compounds containing hydroxyl
groups, and aliphatic or alicyclic amines.
Thus, stabilization of a serine protease using inhibitors has been
described (for example US 2001/0033837 and JP 2004191367, supra).
The problem with this approach is that the inhibitor strongly
diminishes the effect of the enzyme, if it is not removed prior to
use of the preparation. If a potent inhibitor is used, most of the
enzymatic activity is lost. A better approach is to use a
reversible inhibitor of intermediate strength. However, even in
this case, a considerable part of the initial enzymatic activity
will be lost as concentration of the inhibitor is increased in
order to get a good stabilization effect.
DISCLOSURE OF THE INVENTION
It is therefore an object of the present invention to accomplish a
serine protease composition, which is stable in solution and
retains a degree of enzymatic activity which is sufficient for
practical use of the composition.
It is another object of the present invention to provide a serine
protease composition, which is amenable to direct use without prior
steps of preparation from deep frozen or lyophilized material.
It is a further object of the present invention to enable practical
use of reversible inhibitors of serine proteases for stabilization
purposes, through the provision of an additional stabilizing
component.
These, and other objects apparent from the present text, are
attained by the different aspects of the present invention as
claimed.
Thus, one aspect of the invention provides a stabilized serine
protease composition comprising a) a serine protease; b) a
reversible inhibitor of said serine protease; and c) a stabilizing
agent M having the formula I:
##STR00002## wherein
n is 0, 1 or 2;
X is O, N or CH.sub.2;
R.sup.1-R.sup.4 are the same or different, and selected from H,
--CH.sub.2--R.sup.6, --CH.sub.2--O--R.sup.6,
--CH.sub.2--S--R.sup.6, --CH.sub.2--NH--R.sup.6, --CO--O--R.sup.6,
--CO--NH--R.sup.6, --CH.sub.2--NH--CO--R.sup.6,
--CH.sub.2--O--CO--R.sup.6, --CH.sub.2--NH--CO--NHR.sup.6,
--CH.sub.2--NH--CO--OR.sup.6, --CH.sub.2--NH--CS--NHR.sup.6 and
--CH.sub.2--O--CO--NHR.sup.6;
R.sup.5 is as R.sup.1-R.sup.4 or P-Q;
P is selected from --(CH.sub.2).sub.m-- and
--(CH.sub.2).sub.m--Y--(CH.sub.2).sub.m--, wherein m is 1-6 and Y
is O, NH or S;
Q is selected from H, --SO.sub.3, --COOH, --NH.sub.2, --OH and
--CONH.sub.2;
each R.sup.6 individually being selected from H, substituted or
non-substituted lower alkyl, substituted or non-substituted
cycloalkyl, substituted or non-substituted benzyl, substituted or
non-substituted aryl or mono-, bi-, or tricyclic unsubstituted or
substituted heteroaromatic ring(s) with one or more heteroatoms and
non-aromatic heterocycles, the substituents of the substituted
groups being selected from lower alkyl, halogens, substituted or
non-substituted aryl, substituted or non-substituted
hetero-aromatic compounds, non-aromatic heterocycles, alkyloxy,
alkylamino;
or a pharmaceutically acceptable salt thereof.
The present invention derives from initial results from a study on
the stability of thrombin, in which it was surprisingly found that
the inventive combination of a reversible inhibitor of the enzyme
and a stabilizing agent M as defined above had a strong stabilizing
effect on the enzyme in solution. Both the thrombin inhibitor and
the stabilizing agent M alone had stabilization effects on
thrombin, but the combination was several fold better than any of
them (see Example 1). Thus, when a low concentration of enzyme
inhibitor was combined with morpholine, MOPS or related compounds,
a very strong stabilizing effect on the enzyme was obtained. Some
tested compositions were stable, as indicated by less than 30%
decrease in activity, for more than 2 months at 37.degree. C. This
would, according to data in prior publications and confirmed by the
present inventors, correspond to 6 months at room temperature or
2.5 years at refrigerator temperature. The results from the initial
study were expanded to include experiments on other serine
proteases, and in these experiments the surprising stabilizing
effect was also observed.
As exemplified below, the composition according to the invention
exhibits a substantially improved stability as compared to enzyme
compositions without the inventive combination of ingredients b)
and c). With the inventive approach, a low concentration of serine
protease inhibitor may be used, and a satisfactory degree of
stabilization still obtained. For example, the concentration of the
inhibitor may be lower than what has been suggested previously, e g
in US 2001/0033837. With such a low concentration of inhibitor,
much more of the enzymatic activity is retained in the stabilized
enzyme solution.
It should be noted that the increase in stabilization due to the
combination of the reversible serine protease inhibitor and
stabilizing agent M is not regarded as an additional inhibitory
effect provided by M. In fact M, as described in Illustrative
Example A, may lack any serine protease inhibiting capacity.
Without wishing to be bound by theory, the present inventors
believe that the surprisingly increased stabilizing effect observed
is achieved through a beneficial synergy between reversible serine
protease inhibitors and stabilizing agents M of the inventive
composition. The present invention provides such a combination of a
reversible serine protease inhibitor and stabilizing agent M in a
stabilized serine protease composition and use of such a
combination for stabilizing a serine protease composition.
In an embodiment of the invention, the serine protease in the
composition is selected from the group consisting of trypsin,
kallikrein, thrombin, plasmin, urokinase, tissue plasminogen
activator, active form of factor IX, active form of factor X and
active form of factor XI. In a more specific embodiment, the serine
protease is thrombin. In another specific embodiment, the serine
protease is plasmin. In yet another specific embodiment, the serine
protease is trypsin.
Reversible inhibitors to serine proteases are known to persons of
skill in the art, and which one is the optimal to use will vary
depending on what specific serine protease is used. In general, it
is of importance for the intended effect that the inhibitor is not
of great strength. In other words, the inhibitory effect has to be
moderate enough that the enzymatic activity remains usefully high.
As a guideline, it has been found that inhibitors having a K.sub.i
of between 0.01 mM and 2 mM are suitable for use in the composition
according to the invention, with from 0.04 mM to 0.5 mM as a
preferred range.
In one embodiment, in which the serine protease is thrombin, the
reversible inhibitor may be selected from
N-(2'-phenoxy)-4-aminopyridin and derivatives thereof, benzamidine,
N,N-diethylethylenediamine, aminobenzamidine, amidinopyridin and
tert-butylamidin. In another embodiment, in which the serine
protease is thrombin, the reversible inhibitor is selected from
N-(2'-phenoxy)-4-aminopyridin and derivatives thereof,
N,N-diethylethylenediamine, amidinopyridin and tert-butylamidin. In
a more specific embodiment, in which the serine protease is
thrombin, the reversible inhibitor is N-(2'-phenoxy)-4-aminopyridin
or a derivative thereof. In another embodiment, in which the serine
protease is plasmin, the reversible inhibitor is selected from
N,N-diethylethylenediamine, aminobenzamidine and benzamidine. In
another embodiment, in which the serine protease is trypsin, the
reversible inhibitor is selected from aminobenzamidine and
benzamidine. These combinations of enzymes and inhibitors are
illustrative examples, and are not to be interpreted as
limiting.
In one embodiment of the invention, the value of n in formula I is
1 or 2. In a more specific embodiment, n in formula I is 1.
The composition according to the invention comprises a stabilizing
agent M with the general formula I given above. In embodiments of
the invention, stabilizing agent M is a compound of formula II:
##STR00003## wherein
R.sup.1-R.sup.4 are the same or different, and selected from H,
--CH.sub.2--R.sup.6;
R.sup.5 is as R.sup.1-R.sup.4 or P-Q;
P is selected from --(CH.sub.2).sub.m-- and
--(CH.sub.2).sub.m--Y--(CH.sub.2).sub.m--, wherein m is 1-6 and Y
is O, NH or S;
Q is selected from H, --SO.sub.3, --COOH, --NH.sub.2, --OH and
--CONH.sub.2;
each R.sup.6 individually being selected from H, substituted or
non-substituted lower alkyl, substituted or non-substituted
cycloalkyl, substituted or non-substituted benzyl, substituted or
non-substituted aryl or mono-, bi-, or tricyclic unsubstituted or
substituted heteroaromatic ring(s) with one or more heteroatoms and
non-aromatic heterocycles, the substituents of the substituted
groups being selected from lower alkyl, halogens, substituted or
non-substituted aryl, substituted or non-substituted
hetero-aromatic compounds, non-aromatic heterocycles, alkyloxy,
alkylamino;
or a pharmaceutically acceptable salt thereof.
Accordingly, in some embodiments, stabilizing agent M is a compound
of formula III:
##STR00004## wherein
R.sup.5 is --CH.sub.2--R.sup.6 or P-Q;
P is selected from --(CH.sub.2).sub.m-- or
--(CH.sub.2).sub.m--Y--(CH.sub.2).sub.m--, wherein m is 1-6 and Y
is O, NH or S;
Q is selected from H, --SO.sub.3, --COOH, --NH.sub.2, --OH and
--CONH.sub.2.
each R.sup.6 individually being selected from substituted or
non-substituted lower alkyl, substituted or non-substituted
cycloalkyl, substituted or non-substituted benzyl, substituted or
non-substituted aryl, the substituents of the substituted groups
being selected from lower alkyl, halogens, substituted or
non-substituted aryl, substituted or non-substituted
hetero-aromatic compounds, non-aromatic heterocycles, alkyloxy,
alkylamino;
or a pharmaceutically acceptable salt thereof.
In some embodiments of the invention, stabilizing agent M is
selected from the group consisting of morpholine,
3-(N-morpholino)propanesulfonic acid (MOPS), morpholino butyl
sulphonic acid, morpholino propyl carboxylic acid, morpholino ethyl
alcohol and morpholino ethyl sulphonic acid. Thus, examples of
compounds M for use in the compositions of this aspect of the
invention are morpholine and 3-(N-morpholino)propanesulfonic acid
(MOPS). In a specific embodiment of the invention, stabilizing
agent M is morpholine.
A composition according to the invention which shows the
stabilization effect is one in which the serine protease is
thrombin, the reversible inhibitor is
N-(2'-phenoxy)-4-aminopyridin, and stabilizing agent M is
morpholine.
Another composition according to the invention which shows the
stabilization effect is one in which the serine protease is
thrombin, the reversible inhibitor is
N-(2'-phenoxy)-4-aminopyridin, and stabilizing agent M is
3-(N-morpholino)propanesulfonic acid (MOPS).
Another composition according to the invention which shows the
stabilization effect is one in which the serine protease is
thrombin, the reversible inhibitor is aminobenzamidine, and
stabilizing agent M is morpholine.
Another composition according to the invention which shows the
stabilization effect is one in which the serine protease is
plasmin, the reversible inhibitor is N,N-diethylethylenediamine and
stabilizing agent M is morpholine.
Another composition according to the invention which shows the
stabilization effect is one in which the serine protease is
plasmin, the reversible inhibitor is aminobenzamidine and
stabilizing agent M is morpholine.
In serine protease compositions for topical administration, e g to
a wound site, it has been a problem that the composition can easily
flow or be rinsed from the site where it is applied. In order to
solve this problem, it is possible to add to the enzyme composition
an adhesive polymer, which then serves the purpose of making the
composition more viscous and adherent to skin or wound sites. As an
embodiment of the present invention, such an addition of an
adhesive polymer to the inventive composition may have an
additional unexpected and beneficial effect on its stability. The
addition of a polymer then serves the double purpose of increasing
the viscosity and adhesiveness of the composition, at the same time
as it helps the stabilization of the enzyme even further.
In some embodiments of the invention, the composition further
comprises a viscous and adhesive polymer selected from
polysaccharides and gelatin. Thus, the polymer may for example be a
polysaccharide, such as selected from starch, its derivatives,
cellulose, its derivatives, and mixtures thereof. Specific,
non-limiting examples of starches useful as additives to the
composition according to the invention include corn starch and
potato starch and mixtures thereof, whereas non-limiting examples
of useful cellulose derivatives are carboxymethyl cellulose and
ethyl hydroxyethyl cellulose and mixtures thereof. In a specific
embodiment the polysaccharide is carboxymethyl chitosan. In further
embodiments of the invention, said polysaccharide is present in a
concentration of 0.1-5%. However, it is also envisaged that the
polymer is gelatin, such as gelatin from a cold water fish. In some
embodiments of the invention, said gelatin is present in a
concentration of 0.5-20%.
In one embodiment of the invention, said serine protease is present
in a concentration of 0.001-2 mg/ml. In a more specific embodiment,
said serine protease is present in a concentration of 0.01-1
mg/ml.
In one embodiment of the invention, in which the serine protease is
thrombin, the concentration of thrombin is between 5-3500 activity
units/ml.
In one embodiment of the invention, in which the serine protease is
thrombin, the concentration of thrombin is between 200-1000
activity units/ml.
In one embodiment of the invention, in which the serine protease is
thrombin, the concentration of thrombin is between 5-20 activity
units/ml.
In one embodiment of the invention, said reversible inhibitor of
said serine protease is present in a concentration of 0.1-10 mM. In
a more specific embodiment, said reversible inhibitor of said
serine protease is present in a concentration of 0.5-2 mM.
In one embodiment of the invention, said stabilizing agent M is
present in a concentration of 0.02-0.5 M. In a more specific
embodiment, said stabilizing agent M is present in a concentration
of 0.1-0.3 M.
According to another aspect thereof, the present invention provides
use of a composition as described above as a medicament.
Another aspect of the invention concerns use of said composition,
in which the serine protease is thrombin, for the preparation of a
medicament for establishing haemostasis in a subject suffering from
a bleeding. A related aspect of the invention provides a method for
establishing haemostasis in a subject suffering from a bleeding,
comprising applying a composition according to the invention, in
which composition the serine protease is thrombin, to the site of
bleeding in an amount sufficient to diminish or stop said
bleeding.
In connection with such a use or method employing a thrombin
composition according to the invention as a medicament, the
stability of the inventive composition offers benefits in the
circumstances in which it is used. Often, thrombin compositions are
used in the context of emergency situations, wherein it is crucial
to stop subjects from bleeding. In these same situations, the use
of conventional, haemostatic thrombin preparations is difficult,
since they often require cumbersome and time-consuming steps of
thawing (if frozen) and/or dissolution (if lyophilized). The
present invention enables the production of e g such haemostatic
agents in the form of solutions, whose stability is such that they
can readily be stored during extended periods of time, for example
in an ambulance or an emergency helicopter, until needed at the
site of an accident or the like. At this time, they may be used as
is, without any delay due to preparation.
The conventional preparations used to stop bleeding contain fairly
high concentrations of thrombin, between 200-1000 activity
units/ml. In connection with plastic surgery applications, this is
seen as a risk for increased scar formation. Low thrombin
concentration solutions are presently prepared in the clinic by
dilution of concentrated thrombin solutions. No ready to use
preparation is available. Therefore, in a further aspect thereof,
the present invention provides a stabilized thrombin composition
with a considerably lower concentration of thrombin, between 5-20
activity units/ml, and use thereof in plastic surgery.
Another aspect of the invention exploits the known properties of
plasmin, urokinase or tPA as thrombolytic agents. Thus, the
invention provides use of a composition as described above, in
which the serine protease is selected from plasmin, urokinase and
tissue plasminogen activator, for the preparation of a medicament
for thrombolytic treatment. A related aspect provides a method for
thrombolytic treatment in a subject in need thereof, comprising
administering a composition as described above, in which
composition the serine protease is selected from plasmin, urokinase
and tissue plasminogen activator, to the subject in an amount
sufficient for said treatment. In these two related aspects, the
thrombolytic treatment in question may, as non-limiting examples,
be performed in order to treat myocardial infarction or in order to
treat stroke.
As mentioned in the context of the composition aspect of the
invention, the increase in stabilization due to the combination of
the reversible serine protease inhibitor and stabilizing agent M is
not regarded as an additional inhibitory effect provided by M. In
fact M, as described in Illustrative Example A, may lack any serine
protease inhibiting capacity. Without wishing to be bound by
theory, the present inventors believe that the surprisingly
increased stabilizing effect observed is achieved through a
beneficial synergy between reversible serine protease inhibitors
and stabilizing agents M of the inventive composition.
Therefore, in another aspect thereof, the invention provides the
use of a combination of
a) a reversible serine protease inhibitor and
b) a stabilizing agent M of formula I:
##STR00005## wherein
n is 0, 1 or 2;
X is O, N or CH.sub.2;
R.sup.1-R.sup.4 are the same or different, and selected from H,
--CH.sub.2--R.sup.6, --CH.sub.2--O--R.sup.6,
--CH.sub.2--S--R.sup.6, --CH.sub.2--NH--R.sup.6, --CO--O--R.sup.6,
--CO--NH--R.sup.6, --CH.sub.2--NH--CO--R.sup.6,
--CH.sub.2--O--CO--R.sup.6, --CH.sub.2--NH--CO--NHR.sup.6,
--CH.sub.2--NH--CO--OR.sup.6, --CH.sub.2--NH--CS--NHR.sup.6 and
--CH.sub.2--O--CO--NHR.sup.6;
R.sup.5 is as R.sup.1-R.sup.4 or P-Q;
P is selected from --(CH.sub.2).sub.m-- and
--(CH.sub.2).sub.m--Y--(CH.sub.2).sub.m--, wherein m is 1-6 and Y
is O, NH or S;
Q is selected from H, --SO.sub.3, --COOH, --NH.sub.2, --OH and
--CONH.sub.2;
each R.sup.6 individually being selected from H, substituted or
non-substituted lower alkyl, substituted or non-substituted
cycloalkyl, substituted or non-substituted benzyl, substituted or
non-substituted aryl or mono-, bi-, or tricyclic unsubstituted or
substituted heteroaromatic ring(s) with one or more heteroatoms and
non-aromatic heterocycles, the substituents of the substituted
groups being selected from lower alkyl, halogens, substituted or
non-substituted aryl, substituted or non-substituted
hetero-aromatic compounds, non-aromatic heterocycles, alkyloxy,
alkylamino; or a pharmaceutically acceptable salt thereof;
for stabilizing a serine protease composition, wherein the
reversible serine protease inhibitor and the stabilizing agent M
act in synergy to provide a serine protease stabilizing effect.
In this inventive use of a combination of a reversible serine
protease inhibitor and a stabilizing agent for stabilization of a
serine protease composition, the choices of particular components
that may be used and substituents for compounds M are as discussed
above in relation to the composition aspect of the invention.
In yet another aspect, the invention provides a method for the
stabilization of a serine protease, which comprises admixing the
serine protease with a) a reversible inhibitor of said serine
protease; and b) a stabilizing agent M of formula I:
##STR00006## wherein
n is 0, 1 or 2;
X is O, N or CH.sub.2;
R.sup.1-R.sup.4 are the same or different, and selected from H,
--CH.sub.2--R.sup.6, --CH.sub.2--O--R.sup.6,
--CH.sub.2--S--R.sup.6, --CH.sub.2--NH--R.sup.6, --CO--O--R.sup.6,
--CO--NH--R.sup.6, --CH.sub.2--NH--CO--R.sup.6,
--CH.sub.2--O--CO--R.sup.6, --CH.sub.2--NH--CO--NHR.sup.6,
--CH.sub.2--NH--CO--OR.sup.6, --CH.sub.2--NH--CS--NHR.sup.6 and
--CH.sub.2--O--CO--NHR.sup.6;
R.sup.5 is as R.sup.1-R.sup.4 or P-Q;
P is selected from --(CH.sub.2).sub.m-- and
--(CH.sub.2).sub.m--Y--(CH.sub.2).sub.m--, wherein m is 1-6 and Y
is O, NH or S;
Q is selected from H, --SO.sub.3, --COOH, --NH.sub.2, --OH and
--CONH.sub.2;
each R.sup.6 individually being selected from H, substituted or
non-substituted lower alkyl, substituted or non-substituted
cycloalkyl, substituted or non-substituted benzyl, substituted or
non-substituted aryl or mono-, bi-, or tricyclic unsubstituted or
substituted heteroaromatic ring(s) with one or more heteroatoms and
non-aromatic heterocycles, the substituents of the substituted
groups being selected from lower alkyl, halogens, substituted or
non-substituted aryl, substituted or non-substituted
hetero-aromatic compounds, non-aromatic heterocycles, alkyloxy,
alkylamino;
or a pharmaceutically acceptable salt thereof.
In this inventive method for stabilization of a serine protease
composition, the choices of particular components that may be used
and substituents for compounds M are as discussed above in relation
to the composition aspect of the invention.
A further aspect of the invention concerns the use of the
composition as described above for adsorption onto a solid object,
in order that this solid object may provide the enzymatic activity
in question. In particular, it is of interest in many surgical
applications to enter and, in particular, exit arteries while
inflicting as little damage from bleeding as possible. In order to
stop bleeding from an artery, it has previously been suggested to
use a form of "arterial plug" (such objects are also known as
vascular sealing devices, femoral access closure devices (when the
femoral artery is used for entry, e g in angiography), vascular
hemostasis devices and puncture closure devices), for example made
from collagen or another biodegradable material. According to the
present aspect of the invention, such a plug may advantageously be
coated with a composition according to the invention, in which the
serine protease is thrombin. Such a plug achieves faster sealing of
the opening of the artery, in that the thrombin of the composition
aids in blood clotting around the plug. Thus, the invention
provides, in this aspect, a vascular haemostasis device having an
amount of the composition according to the invention, in which the
serine protease is thrombin, adsorbed on it. The vascular
haemostasis device is preferably made from a biodegradable solid or
semi-solid material, such as collagen, chitosan or other biological
polymer.
Another aspect of the invention concerns the novel identification
of N,N-diethylethylenediamine as a serine protease inhibitor. Thus,
in this aspect, the invention provides use of
N,N-diethylethylenediamine as an inhibitor of a serine protease, as
well as a method of inhibiting a serine protease, comprising
admixing therewith an inhibitory amount of
N,N-diethyl-ethylenediamine. In some embodiments of this aspect of
the invention, the serine protease is plasmin. In other embodiments
of this aspect of the invention, the serine protease is
thrombin.
It is generally preferred, for the realization of all the
advantages of the invention's different aspects, that the
composition according to the invention is in a form selected from a
solution and a gel. In this regard, aqueous solutions and aqueous
gels are more preferred.
DEFINITIONS
As used herein, the term "lower alkyl" means an unbranched or
branched, cyclic, saturated or unsaturated (alkenyl or
alkynyl)hydrocarbyl radical which may be substituted or
unsubstituted. Where cyclic, the alkyl group is preferably C3-C12,
more preferably C5-C10, most preferably C5-C7. Where acyclic, the
alkyl group is preferably C1-C10, more preferably C1-C6, more
preferably methyl, ethyl, propyl (n-propyl, isopropyl), butyl
(branched or unbranched) or pentyl, most preferably methyl.
As used herein, the term "aryl" means an aromatic group, such as
phenyl or naphthyl, or a mono-, bi-, or tricyclic heteroaromatic
group containing one or more heteroatom(s) preferably selected from
N, O and S, such as pyridyl, pyrrolyl, quinolinyl, furanyl,
thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl,
triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, imidazolyl,
pyrimidinyl, indolyl, pyrazinyl, indazolyl, pyrimidinyl,
thiophenetyl, pyranyl, carbazolyl, acridinyl, quinolinyl,
benzoimidazolyl, benzthiazolyl, purinyl, cinnolinyl, pterdinyl.
As used herein, the term "functional group" means, in the case of
unprotected: hydroxy-, thiolo-, aminofunction, carboxylic acid, and
in the case of protected: lower alkoxy, N--, O--, S-- acetyl,
carboxylic acid ester.
As used herein, the term "heteroaryl" means an aromatic group
containing one or more heteroatom(s) preferably selected from N, O
and S, such as pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl,
oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl,
triazolyl, imidazolyl, pyrimidinyl, indolyl, pyrazinyl or
indazolyl.
As used herein, the term "non-aromatic heterocycle" means a
non-aromatic cyclic group containing one or more heteroatom(s)
preferably selected from N, O and S, such as a cyclic amino group
such as pyrrolidinyl, piperidyl, piperazinyl, morpholinyl or a
cyclic ether such as tetrahydrofuranyl, monosaccharide.
As used herein, the term "halogen" means fluorine, chlorine,
bromine or iodine.
As used herein, the term "substituted" means that the groups
concerned are substituted with a functional group such as hydroxyl,
amine, sulfide, silyl, carboxylic acid, halogen, aryl, etc.
Examples of pharmaceutically acceptable addition salts for use in
the compositions of the present invention include those derived
from mineral acids, such as hydrochlorid, hydrobromic, phosphoric,
metaphosphoric, nitric and sulphuric acids, and organic acids, such
as tartaric, acetic, citric, malic, lactic, fumaric, benzoic,
glycolic, gluconic, succinic, and arylsulphonic acids.
Pharmaceutically acceptable excipients described herein, for
example, vehicles, adjuvants, carriers or diluents, are well known
to those who are skilled in the art and are readily available to
the public. A pharmaceutically acceptable carrier may be one which
is chemically inert to the active compounds and which have no
detrimental side effects or toxicity under the conditions of use.
Pharmaceutical formulations may be found e g in Remington: The
Science and Practice of Pharmacy, 19.sup.th edition, Mack Printing
Company, Easton, Pa. (1995).
As detailed in the description of the invention, a possible choice
of inhibitor for use in the composition and methods according to
the invention is "N-(2'-phenoxy)-4-aminopyridin and derivatives
thereof". By this is meant a compound having the formula IV:
##STR00007## wherein
R.sup.1 is selected from H, C1-C6-alkyl, C3-C7-cyclo alkyl, phenyl,
benzyl acetyl and benzoyl;
X is selected from oxygen, nitrogen and sulfur;
R.sup.2 and R.sup.3 is each individually selected from H, halogen,
hydroxyl, C1-C6-alkyl, C3-C7-cyclo alkyl, C1-C6-alkyloxy; and
R.sup.4 is selected from H, C1-C6-alkyl, arylalkyl and acyl.
Preferred such inhibitors have the formula V:
##STR00008## wherein
R.sup.1 is selected from C1-C6-alkyl, C3-C7-cyclo alkyl, phenyl and
benzyl;
R.sup.2 and R.sup.3 is each individually selected from H, halogen,
hydroxyl, C1-C6-alkyl, C3-C7-cyclo alkyl and C1-C6-alkyloxy.
EXAMPLES
The following examples illustrate the invention, and are not to be
interpreted as limiting.
In the following description of experiments conducted in accordance
with the present invention, the time it takes to reach 70% of the
initial activity is used as a numerical value for the stability of
an enzyme solution. This value, denoted "T 70%", is chosen since it
corresponds to what could be accepted as a maximum permitted loss
in activity during a life span of a commercial product.
In the experimental studies, a high temperature (37.degree. C.) has
been used, as well as a high concentration of enzyme. This has been
made in order to obtain stability data in reasonably short times,
and not have to wait for months or years. The stability's
dependency on temperature has been studied, and the results given
in Example 1. This study showed that the inactivation process is
about 3 times slower at room temperature, and around 20 times
slower at refrigerator temperature, than the process actually
measured (at 37.degree. C.).
Furthermore, the inactivation process is concentration dependent,
and is more rapid at higher concentrations of the enzyme. The
concentration of thrombin used in Example 1 was 1 mg/ml (or 3300
units/ml), i e higher than the 0.1-0.3 mg/ml used in present
commercially available preparations and/or devices. Studies of the
concentration dependency has shown that the inactivation process is
3-4 times slower at those concentrations as compared to the
concentration used in Example 1.
Taking this together would give a factor of between 10-12, with
which to multiply the T 70% value in order to arrive at what
corresponds to room temperature storage conditions for a commercial
product containing a serine protease, such as a haemostatic
preparation containing thrombin. In Table 1, the composition with
N-(2'-phenoxy)-4-aminopyridine and MOPS has a T 70% of 120 days.
That would correspond to a value, for a 0.1-0.3 mg/ml product, of
more than 1200 days in room temperature conditions, i e more than
three years. This clearly exceeds anything previously accomplished,
regarding the stabilization of thrombin in solution.
Example 1
Stabilization of Human Thrombin
To determine the coagulant activity of thrombin solutions, the time
to clotting of a fibrinogen solution (1.3 mg/ml) after additions of
various dilutions of a solution of a particular human thrombin
(derived from plasma, 3300 units/mg, Biovitrum AB, Sweden) was
measured. The clotting times were measured using an Amelungen Kc 1
coagulometer (Amelungen, Germany). To study the stabilities of
thrombin solutions with different additives, the samples were
incubated in a thermostat chamber kept at 37.degree. C. Aliquots
were taken out at various time intervals, and the remaining
thrombin activity in these aliquots was measured. From the values
obtained, activity decay curves could be constructed.
Activity decay curves of 1 mg/ml thrombin solutions in 10 mM HEPES,
0.13 M NaCl buffer, pH 7.4, showed T 70% values around 1.6 days at
37.degree. C. Corresponding experiments at room temperature (around
21.degree. C.) showed a T 70% value of 5.4 days, whereas after
refrigerator storage (around 5.degree. C.), the T 70% value was 36
days. Thus, as expected, there is a strong temperature
dependency.
Solutions containing 1 mg/ml of thrombin in 10 mM HEPES and 0.13 M
NaCl at pH 7.4 with the indicated stabilizing additive(s) were put
in the thermostat chamber, and their activity decay curves were
determined. The results obtained are shown in Table 1. Data on the
corresponding 1 mg/ml thrombin solution without additives is
included for comparison.
TABLE-US-00001 TABLE 1 Stabilization of thrombin Stabilizing
additive(s) T 70% (days) None 1.6 0.20 M MOPS 7.5 0.20 M morpholine
8.5 0.20 M morpholino butyl sulphonic acid 4.0 0.20 M morpholino
propyl carboxylic acid 4.1 0.20 M morpholino ethyl alcohol 3.8 0.20
M morpholino ethyl sulphonic acid 3.2 0.5 mM aminobenzamidine 20
0.20 M MOPS + 68 0.5 mM aminobenzamidine 3.1 mM
N-(2'-phenoxy)-4-aminopyridin 74 1.9 mM
N-(2'-phenoxy)-4-aminopyridin 35 1.9 mM
N-(2'-phenoxy)-4-aminopyridin + 68 0.5 mM aminobenzamidine 0.20 M
MOPS + 120 1.9 mM N-(2'-phenoxy)-4-aminopyridin 0.20 M
N,N-diethylethylenediamine 10 0.20 M MOPS + 22 0.20 M
N,N-diethylethylenediamine
It is evident that the tested compounds all have a stabilizing
effect. However, there is a synergistic effect of combinations of
inhibitor and morpholine-containing compound in accordance with the
invention, as evidenced by the superior results obtained with such
combinations. As the table above illustrates, the addition of 0.20
M MOPS alone gives an increase in stabilization by a factor of 4.7,
and the addition of 0.5 mM of the reversible thrombin inhibitor
aminobenzamidine gives a stabilization increase by a factor of
12.5. The inventive combination, however, stabilizes the thrombin
composition much better, by a factor of 42.5. The inventive
combination of MOPS and N,N-diethylethylenediamine is also better
at stabilizing the enzyme than the individual components. Likewise,
the combination of 0.20 M MOPS and 1.9 mM
N-(2'-phenoxy)-4-aminopyridin is seen to confer a very high
stabilization increase, a factor of 75, whereas the individual
components increase stability by a factor of 4.7 and 22,
respectively.
The thrombin used in the study is human thrombin derived from
plasma. Recombinant human thrombin has also been studied and has
essentially shown the same behavior.
Example 2
Stabilization of Bovine Thrombin
Stabilization of bovine thrombin was studied. The experimental
setup was the same as in Example 1, but the concentration of bovine
thrombin (Baxter) used was 0.4 mg/ml. Upon storage at 37.degree.
C., the thrombin solution in HEPES buffer showed a T 70% value of
1.3 days. The thrombin solution in HEPES buffer plus 3.0 mM
N-(2'-phenoxy)-4-aminopyridin and 0.20 M MOPS showed a T 70% value
of 54 days.
The results obtained show that bovine thrombin is somewhat more
labile than the preparations of human thrombin studied, but that a
very good stabilizing effect is nevertheless obtained by the
compositions of the invention.
Example 3
Low Concentrations of Thrombin
Stabilization of thrombin in compositions containing low
concentrations of thrombin was studied. The stabilizing effect of
the compositions according to the invention was demonstrated to
work also for comparatively low concentrations of thrombin.
A 15.0 activity units/ml solution of human thrombin (derived from
plasma, 3300 units/mg, Biovitrum AB, Sweden) in HEPES buffer, pH
7.4, showed a T 70% value of 23 days. The corresponding solution in
HEPES buffer, pH 7.4, plus 2.0 mM N-(2'-phenoxy)-4-aminopyridin and
0.20 M MOPS exhibited a T 70% value of 92 days.
Example 4
Stabilization of Plasmin
The stabilization of solutions of plasmin in accordance with the
invention was tested. The activity of plasmin was determined using
chromogenic peptide substrate Chromozym TH (Pentapharm,
Switzerland) and measurement of absorbance change at 405 nm in a
spectrophotometer. Solutions containing 100 .mu.g/ml of plasmin
(specific activity 3.2 units/mg, Sigma-Aldrich) in 10 mM HEPES and
0.13 M NaCl, pH 7.4, as well as stabilizers as indicated in Table 2
below were incubated at 37.degree. C., and samples were taken out
at various time intervals for activity determination. The results
obtained are shown in Table 2.
TABLE-US-00002 TABLE 2 Stabilization of plasmin Stabilizing
additive(s) T 70% (hours) None 3 0.20 M morpholine 12 0.13 M
N,N-diethylethylenediamine 8 1 mM benzamidine 16 1.3 mM
aminobenzamidine 52 0.20 M morpholine + 22 0.13 M
N,N-diethylethylenediamine 0.20 M morpholine + 216 1.3 mM
aminobenzamidine
From these results, it is evident that a very strong stabilization
is obtained using the combination in accordance with the invention.
0.20 M morpholine increases the stability of the plasmin
composition by a factor of 4, 0.13 M N,N-diethylethylenediamine by
a factor of 2.7 and 1.3 mM aminobenzamidine by a factor of 17.
However, the combination of morpholine and
N,N-diethylethylenediamine increases the stability of the plasmin
composition by a factor of 7.3, and the combination of morpholine
and aminobenzamidine increases the stability by a factor of 72.
Example 5
Stabilization of Trypsin
The stabilization of solutions of trypsin in accordance with the
invention was tested. The activity of trypsin was determined using
tosyl arginine methyl ester (TAME) as substrate and measuring the
absorbance change at 247 nm in a spectrophotometer. Solutions
containing 100 .mu.g/ml of trypsin (TPCK-treated, Sigma-Aldrich) in
10 mM HEPES and 0.13 M NaCl, pH 7.4, as well as stabilizers as
indicated in Table 3 below were incubated at 37.degree. C., and
samples were taken out at various time intervals for activity
determination. The results obtained are shown in Table 3.
TABLE-US-00003 TABLE 3 Stabilization of trypsin Stabilizing
additive(s) T 70% (hours) None 0.6 0.5 M morpholine 8 1 mM
benzamidine 43 0.5 M morpholine + 88 1 mM benzamidine
Again, the stabilizing effect is the greatest in the composition
according to the invention. Thus, 0.5 M morpholine alone gives an
increase in stabilization by a factor of 13, and 1 mM benzamidine
alone gives an increase in stabilization by a factor of 72. The
inventive combination, on the other hand, gives an increase in
stabilization by a factor of 147.
Example 6
Stabilization of Thrombin with CMC
Tests of thrombin solutions containing between 1.0 and 2.0%
carboxymethyl cellulose (CMC) for their adhesiveness to human skin
showed that the addition of CMC increased both viscosity and
adhesiveness strongly. Surprisingly, however, it was also found
that the stability of these thrombin solutions was further
increased. A 1 mg/ml human thrombin (derived from plasma, 3300
units/mg, Biovitrum AB, Sweden) solution in 0.5 mM
aminobenzamidine, 0.20 M MOPS, 10 mM HEPES, 0.13 M NaCl being 2.0%
with respect to CMC was incubated at 37.degree. C., and the
activity decay curve was determined. The T 70% value obtained was
175 days.
Example 7
Stabilization with Other Adhesive Polymers
Four other polymers were also studied: ethyl hydroxyethyl cellulose
(EHEC), potato starch, corn starch and cold water fish gelatin. All
four of these polymers increased the adhesiveness and viscosity of
thrombin solutions. The compatibility and stability of thrombin
solutions with the polymers were further studied by incubation at
37.degree. C. of 1 mg/ml human thrombin (derived from plasma, 3300
units/mg, Biovitrum AB, Sweden) solutions in 0.20 M MOPS, 0.5 mM
aminobenzamidine, 10 mM HEPES, 0.13 M NaCl, pH 7.4, containing the
various polymers. The concentrations of the polymers used were:
EHEC, 0.6%; the two different starches, 4.0%; and gelatin, 12.8%.
EHEC was fully compatible with thrombin, and the same T 70% value,
i.e. around 65 days, was obtained as with the corresponding
solution without EHEC. The starch containing solutions had T 70%
values of 22 and 26 days. The stability of thrombin was very good
in gelatin with a T 70% value of more than 90 days, which
demonstrates additional stabilizing effect of cold water fish
gelatin.
Example 8
Bleeding Experiments
The ability of inventive compositions to stop bleeding was tested
in a series of experiments on rabbits. The model chosen was
incisions in the liver which is a frequently used model. The
abdomen of the rabbit was opened and the liver exposed.
Standardized cuts of 3 mm length were made in the liver surface and
an 0.10 ml amount of test solution was applied to the wound using a
syringe. The time to haemostasis was measured. 10-12 experiments
were performed with each solution. An average value of bleeding
time was calculated after removal of the highest and the lowest
value in each series of experiments. For comparison, the commonly
used haemostatic agent Tisseel (Baxter), a fibrin glue, was also
included in the study. Tisseel was used essentially according to
the manufacturer's recommendations. 0.2 ml of solution was applied
to each wound using a double syringe with a mixing chamber. The
results obtained are given in Table 4 below.
TABLE-US-00004 TABLE 4 Bleeding experiments Average bleeding
Composition time (s) 10 mM HEPES, 0.13 M NaCl, pH 7.4 106 10 mM
HEPES, 0.13 M NaCl, pH 7.4 + 65 1.5% CMC 10 mM HEPES, 0.13 M NaCl,
pH 7.4 + 31 1.5% CMC + 0.20 M MOPS + 1000 units/ml thrombin 10 mM
HEPES, 0.13 M NaCl, pH 7.4 + 26 2 mM aminobenzamidine + 0.20 M MOPS
+ 1000 units/ml thrombin Tisseel (Baxter) 0.2 ml solution 31
As is evident from these results, the thrombin solution stabilized
according to the invention is the most effective in quickly
establishing haemostasis in a bleeding subject, comparable to or
better than a commonly used agent.
Example 9
Compatibility with Porous Materials
A solution containing 0.4 mg/ml human thrombin (derived from
plasma, 3300 units/mg, Biovitrum AB, Sweden) in 10 mM HEPES, 0.14 M
NaCl, 0.5 mM aminobenzamidine, 0.20 M MOPS of pH 7.4 was adsorbed
into a piece of polyurethane plaster (marketed as Ligasone by
Hartmann Scandicare AB, Anderstorp, Sweden). An amount of solution
sufficient to saturate the polyurethane piece was used. The piece
was transferred to a tube, which was then closed to prevent
evaporation. The tube was kept at 37.degree. C., and samples of
solution were taken out at intervals by a slight pressure on the
polyurethane piece. The activity decay curve showed a T 70% value
of 74 days, corresponding to a stability increase by a factor of
46.
Example 10
Adsorption of Enzyme onto a Solid Phase
Adsorption of thrombin in stabilizing solutions to surfaces was
tested. Solid flakes of chitosan (at least 85% deacetylated,
Sigma-Aldrich), around 3.times.3 mm, were incubated for 10 minutes
in solutions of 400 units/ml human thrombin (derived from plasma,
3300 units/mg, Biovitrum AB, Sweden) in 10 mM HEPES, 0.13 M NaCl,
pH 7.4. The solutions had the following additions: 1) none, 2) 0.10
M morpholine, 2 mM N-(2'-phenoxy)-4-aminopyridine, 3) 0.10 M
morpholine, 2 mM N-(2'-phenoxy)-4-aminopyridine, 0.5% carboxymethyl
cellulose. The flakes were then taken up and dried on filter paper.
To get a measure of thrombin clotting activity, a flake was put in
a test tube and 0.4 ml of 1.3 mg/ml fibrinogen solution was added.
To improve clot detection, the tube also contained a small steel
ball. The clotting times obtained initially on flakes from the
various incubation mixtures varied between 1 to 4 minutes. After
incubation in Eppendorf tubes at 37.degree. C. for 7 days, the
clotting times for flakes incubated in solution 1) were strongly
prolonged. The values were between 24 and 27 minutes. In contrast,
the clotting times for flakes incubated in solutions 2) and 3) were
in the range of from 1 to 2.5 minutes, i e the same as the starting
values. Evidently, a strong stabilization of thrombin activity is
obtained by using solutions 2) and 3). To test the in vivo
haemostatic activity chitosan flakes incubated in solution 3) were
applied to wounds in rabbit liver according to the animal model
described in Example 8. The average time to haemostasis was 27
seconds (based on six experiments).
Illustrative Example A
Morpholine is not a Thrombin Inhibitor
The possibility that morpholine is an inhibitor of thrombin was
evaluated. The fibrinogen clotting activity of thrombin is usually
measured by clotting tests, wherein the time to coagulation of a
fibrinogen solution is detected by mechanical or optical devices.
The clotting tests in this experimental setup were performed in
0.01 M HEPES, 0.13 M NaCl buffer of pH 7.4, which is according to
standard procedure (EU Pharmacopeia). A human thrombin (derived
from plasma, 3300 units/mg, Biovitrum AB, Sweden) solution
containing 89 units/ml was used and dilutions of 1/5, 1/10 and 1/16
were tested. Solutions of various concentrations of morpholine were
prepared in the HEPES buffer by adding a concentrated morpholine
solution adjusted to pH 7.4. When morpholine is dissolved in water,
the pH goes up to 9-10, so HCl was added to get a pH of 7.4. That
also increased the ionic strength of the solution. Table I shows
the results obtained. Observed clotting times were converted to
concentrations of thrombin using a standard curve.
TABLE-US-00005 TABLE 5 Inhibitory effect of morpholine Apparent
Morpholine NaCl added Apparent thrombin inhibition concentration
(M) (M) concentration (U/ml) (%) 0 0 89 0 0.05 0 87 4 0.10 0 71 20
0.15 0 49 45 0.20 0 51 43 0 0.05 74 17 0 0.10 53 40 0 0.15 58 34 0
0.20 55 37
As evident from these results, there was a prolongation of clotting
times with increasing concentration of morpholine up to a certain
level. However, the same thing was observed when the ionic strength
was increased with NaCl, and a similar profile was exhibited. Thus,
the prolongation effect was, in all likelihood, caused by the
increase in ionic strength. Further, it is known that an increase
of ionic strength from 0.15 M to 0.22 M causes a change in the
polymerization of fibrin (B. Blomback, Thrombosis Research, vol.
83, (1996), p. 1-75, especially p. 18). This actually corresponds
to the range studied in this experimental series, in which the
initial concentration of NaCl was 0.13 M and then increased to 0.18
M and onwards up to 0.33 M. This also corresponds to the plateau
level observed. In conclusion, morpholine itself is not an
inhibitor of thrombin.
* * * * *